The biosynthetic routes in Pseudomonas aeruginosa to the following hexuronic acid derivatives will be investigated: 2,3-diacetamido-2,3-dideoxy-D-glucuronic acid, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (Man(NAc)2U), and 2,3-diacetamido-2,3-dideoxy-L-guluronic acid (Gul(NAc)2U). These amino uronic acids are found in the O-specific polysaccharides of certain strains of Pseudomonas aeruginosa. A derivative of Man(NAc)2U occurs together with Gul(NAc)2U in the same O antigen chain. Sugar nucleotides botained from P. aeruginosa will be fractionated and the nucleoside diphosphate (NDP) amino and diaminohexuronic acids will be isolated and characterized. In each case an attempt will also be made to isolate and identify the precursors of the NDP diaminohexuronic acids. NDP-glycose precursors radiolabeled in the glycosyl moiety will be incubated with crude enzyme extracts of the appropriate strain of P. aeruginosa, the reaction mixtures will be subjected to paper chromatographic and electrophoretic separation, and reaction products will be isolated and identified to the extent possible. Subsequent larger scale studies using partially purified enzymes will be aimed toward delineating each of the steps in the biosynthesis. Although P. aeruginosa is usually an innocuous organism, it can pose a serious health threat to individuals whose normal health status is altered by medical treatment or concurrent illness. The lipopolysaccharides of P. aeruginosa are of particular interest due to their role as immunogens. Little structural or biosynthetic information concerning these lipopolysaccharides is at present available, due in part to the presence of unusual features like aminohexuronic acids. Work described in this proposal is designed to furnish such information. This research should furnish information to aid in fulfilling the longer term objectives of understanding biosynthetic routes to carbohydrate moities and the involvement of the latter in the formation of complex polysaccharides.